Printed in Sweden Copyright 0 1979 by Academic Press, Inc. All rights of reproduction in any form reserved 0014-4827/79/@80283-07$02.00/0
Experimental Cell Research 121 (1979) 283-289
RELATIONSHIP BIOSYNTHESIS CONCANAVALIN
BETWEEN
PHOSPHATIDYLCHOLINE
AND CELLULAR A-STIMULATED
COMMITMENT
IN
LYMPHOCYTES
S. SHI-HUA CHEN Pathology Department, Stanford University, and Palo Alto Veterans Administration Hospital, Stanford, CA 94305, USA
SUMMARY The incorporation of [*4C]choline into phosphatidylcholine was studied in lymphocyte cultures exposed to concanavalin A (ConA). The lectin was found to induce an increase in the incorporation of the label with following features: 1. It occurs very promptly after exposure. 2. It is not elicited by a non-mitogenic lectin. 3. The increase in the early stage is proportional to lectin concentration. 4. It can be terminated by a competitive inhibitor of ConA binding. 5. The extent of the increase shows a correlation with the rate of cellular commitment to initiate DNA synthesis. These results suggest that in the mitogenic stimulation of T lymphocytes enhanced synthesis of membrane phospholipids is a precommitment event.
In recent years evidence from studies with cultured cells has accumulated in favor of a commitment point (restriction point) between GO and S in the cell cycle [l]. Presumably, once the biochemical events associated with the commitment point have occurred a cell is irreversibly committed to initiate DNA synthesis. Lymphocytes are normally quiescent cells until triggered by either mitogens or antigens. The initial step of lymphocyte activation by concanavalin A (ConA) is the binding of lectin to the surface receptors. After a period of induction, responsive cells become committed to mitogenesis. Committed lymphocytes will go on to initiate DNA synthesis even if the mitogen is removed from the cells [2]. In this response lymphocyte activation by ConA presents an experimental model for identifying the biochemical events associated with the commitment point. 19-791809
A previous study by Cornell et al. [3] with cultured cell lines showed that when cells were grown in a lipid-depleted culture medium, selective inhibition of the synthesis of phosphatidylcholine or of other lipids led to a Gl cell cycle arrest. The arrest could be reversed by restoring the synthetic pathways or their lipid products. Chen et al. [4] reported that treatment of phytohemagglutinin (PHA)-stimulated lymphocytes with an inhibitor of sterol synthesis prevented both the increase in sterol synthesis and the increase in DNA synthesis. These observations suggest a coordination between lipid metabolism and the onset of DNA synthesis during cell proliferation. Phosphatidylcholine is a major constituent of cell membranes. During the mitogenic stimulation of lymphocytes synthesis de novo of phosphatidylcholine increases very promptly [5, 6, 71. This present study atExp Cell Res 121 (1979)
284
S. Shi-Hua Chen 7
b
Fig. 1. Abscissa: time after ConA addition (hours); ordinate: [‘Qholine incorporation (a) cpm X lo-*; (b) cpm X 10-3. ConA effect on the kinetics of [‘4C]choline incorporation into lipids. Lymphocytes (1aScells/tube) were cultured with m---m, 0; CO, 25; or A---A, 1.50 &ml of ConA. At different times the synthesis of phosphatidylcholine was monitored with either (a) l-h pulses of 2 @Zi of [‘4C]choline or (b) 3-h pulses of 0.8 &i of [“C]choline. The incorporation of ‘y3 into the total lipid fraction was measured. Data are plotted as the niean of duplicate tubes at the mid-point of the pulse.
tempts to determine whether there is a relationship between enhanced phosphatidylcholine synthesis and the commitment of a responsive lymphocyte to initiate DNA synthesis. MATERIAL
AND METHODS
Reagents ConA (Sigma Chemical Co., St Louis, MO) and wheat germ agglutinin (WGA) (Sigma Chemical Co.) were dissolved in Dulbecco’s phosphate-buffered saline (PBS), stored at 4 “C and used within 7 days. a-Methylo-rnannoside @MM) (GIBCO, Grand Island, N.Y.) was dissolved in RPMI-1640 medium as a 1 M solution and added to cultures to a final concentration of 0.1 M. ConA, WGA and aMM were sterilized by Mill&e filtration. [MethylJVjcholine chloride (55 mCi/mM in ethanol solution, New England Nuclear, Boston, Mass.) was diluted 1 : 5 with PBS. It was added to culture tubes in volume of either 20 ~1 (0.8 &i) or 50 ~1 (2 &i). [Methyl-SI$]thymidine (2 Ci/ mM, New England Nuclear) was diluted with RPMI1640, and added to cultures in a volume of 10 ~1. RPMI-1640 medium, Dulbecco’s PBS, gentamicin, glutamine and fetal calf serum (FCS) were purchased from Microbiological Associates (Bethesda, MD).
Lymphocytes cultures Peripheral blood lymphocytes were isolated from heparinized venous blood, obtained from healthy hu-
man donors, by the Ficoll-Hypaque method [S]. Lymphocytes were cultured in complete media (RPMI-1640 supplemented with 2 mM glutamine, 0.1 mg/ml gentamicin and 15% FCS). In the [‘4Clcholine incorporation studies, lymphocytes were cultured in plastic-tubes at a cell density of 1x l@ cell/tube in a volume of 0.5 ml. In the [3H]thymidine incorporation studies, lymphocytes were cultured in a microculture system, using flat bottom sterile tissue culture plates, at cell density of 0.2~ 106 cell/well in a volume of 0.1 ml. Lymphocyte cultures were incubated at 37°C in a humidified atmosphere of 5% CO, in air. They were incubated for 24 h before the addition of ConA.
Measurement of [‘4C]choline incorporation into the total lipid fraction At various times after ConA addition. Ivmnhocvtes were pulsed with either 0.8 &i of [14C]chbli
Experimental Cell Research 121 (1979) 283-289
RELATIONSHIP BIOSYNTHESIS CONCANAVALIN
BETWEEN
PHOSPHATIDYLCHOLINE
AND CELLULAR A-STIMULATED
COMMITMENT
IN
LYMPHOCYTES
S. SHI-HUA CHEN Pathology Department, Stanford University, and Palo Alto Veterans Administration Hospital, Stanford, CA 94305, USA
SUMMARY The incorporation of [*4C]choline into phosphatidylcholine was studied in lymphocyte cultures exposed to concanavalin A (ConA). The lectin was found to induce an increase in the incorporation of the label with following features: 1. It occurs very promptly after exposure. 2. It is not elicited by a non-mitogenic lectin. 3. The increase in the early stage is proportional to lectin concentration. 4. It can be terminated by a competitive inhibitor of ConA binding. 5. The extent of the increase shows a correlation with the rate of cellular commitment to initiate DNA synthesis. These results suggest that in the mitogenic stimulation of T lymphocytes enhanced synthesis of membrane phospholipids is a precommitment event.
In recent years evidence from studies with cultured cells has accumulated in favor of a commitment point (restriction point) between GO and S in the cell cycle [l]. Presumably, once the biochemical events associated with the commitment point have occurred a cell is irreversibly committed to initiate DNA synthesis. Lymphocytes are normally quiescent cells until triggered by either mitogens or antigens. The initial step of lymphocyte activation by concanavalin A (ConA) is the binding of lectin to the surface receptors. After a period of induction, responsive cells become committed to mitogenesis. Committed lymphocytes will go on to initiate DNA synthesis even if the mitogen is removed from the cells [2]. In this response lymphocyte activation by ConA presents an experimental model for identifying the biochemical events associated with the commitment point. 19-791809
A previous study by Cornell et al. [3] with cultured cell lines showed that when cells were grown in a lipid-depleted culture medium, selective inhibition of the synthesis of phosphatidylcholine or of other lipids led to a Gl cell cycle arrest. The arrest could be reversed by restoring the synthetic pathways or their lipid products. Chen et al. [4] reported that treatment of phytohemagglutinin (PHA)-stimulated lymphocytes with an inhibitor of sterol synthesis prevented both the increase in sterol synthesis and the increase in DNA synthesis. These observations suggest a coordination between lipid metabolism and the onset of DNA synthesis during cell proliferation. Phosphatidylcholine is a major constituent of cell membranes. During the mitogenic stimulation of lymphocytes synthesis de novo of phosphatidylcholine increases very promptly [5, 6, 71. This present study atExp Cell Res 121 (1979)
284
S. Shi-Hua Chen 7
b
Fig. 1. Abscissa: time after ConA addition (hours); ordinate: [‘Qholine incorporation (a) cpm X lo-*; (b) cpm X 10-3. ConA effect on the kinetics of [‘4C]choline incorporation into lipids. Lymphocytes (1aScells/tube) were cultured with m---m, 0; CO, 25; or A---A, 1.50 &ml of ConA. At different times the synthesis of phosphatidylcholine was monitored with either (a) l-h pulses of 2 @Zi of [‘4C]choline or (b) 3-h pulses of 0.8 &i of [“C]choline. The incorporation of ‘y3 into the total lipid fraction was measured. Data are plotted as the niean of duplicate tubes at the mid-point of the pulse.
tempts to determine whether there is a relationship between enhanced phosphatidylcholine synthesis and the commitment of a responsive lymphocyte to initiate DNA synthesis. MATERIAL
AND METHODS
Reagents ConA (Sigma Chemical Co., St Louis, MO) and wheat germ agglutinin (WGA) (Sigma Chemical Co.) were dissolved in Dulbecco’s phosphate-buffered saline (PBS), stored at 4 “C and used within 7 days. a-Methylo-rnannoside @MM) (GIBCO, Grand Island, N.Y.) was dissolved in RPMI-1640 medium as a 1 M solution and added to cultures to a final concentration of 0.1 M. ConA, WGA and aMM were sterilized by Mill&e filtration. [MethylJVjcholine chloride (55 mCi/mM in ethanol solution, New England Nuclear, Boston, Mass.) was diluted 1 : 5 with PBS. It was added to culture tubes in volume of either 20 ~1 (0.8 &i) or 50 ~1 (2 &i). [Methyl-SI$]thymidine (2 Ci/ mM, New England Nuclear) was diluted with RPMI1640, and added to cultures in a volume of 10 ~1. RPMI-1640 medium, Dulbecco’s PBS, gentamicin, glutamine and fetal calf serum (FCS) were purchased from Microbiological Associates (Bethesda, MD).
Lymphocytes cultures Peripheral blood lymphocytes were isolated from heparinized venous blood, obtained from healthy hu-
man donors, by the Ficoll-Hypaque method [S]. Lymphocytes were cultured in complete media (RPMI-1640 supplemented with 2 mM glutamine, 0.1 mg/ml gentamicin and 15% FCS). In the [‘4Clcholine incorporation studies, lymphocytes were cultured in plastic-tubes at a cell density of 1x l@ cell/tube in a volume of 0.5 ml. In the [3H]thymidine incorporation studies, lymphocytes were cultured in a microculture system, using flat bottom sterile tissue culture plates, at cell density of 0.2~ 106 cell/well in a volume of 0.1 ml. Lymphocyte cultures were incubated at 37°C in a humidified atmosphere of 5% CO, in air. They were incubated for 24 h before the addition of ConA.
Measurement of [‘4C]choline incorporation into the total lipid fraction At various times after ConA addition. Ivmnhocvtes were pulsed with either 0.8 &i of [14C]chbli
